Iron is an essential nutrient for the growth of Escherichia coli K12, and the tonB gene is an essential component of all high affinity iron transport systems in E.coli. The TonB protein has an apparent molecular weight of 36,000 daltons in denaturing polyacrylamide gels; it is located in the cell envelope fraction; the details of its role in iron transport remain an enigma. Recombinant plasmids carrying the tonB gene have been constructed and the DNA sequence of the tonB gene determined. The tonB promoter and transcription terminator have been deduced from S1-nuclease mapping of tonB RNA. Preliminary experiments suggest that transcription of tonB is elevated under conditions of iron limitation. The specific aims of this proposal are (i) to purify the TonB protein to homogeneity, (ii) to determine the NH2-terminal amino acid sequence of TonB protein, (iii) to determine the biochemical half-life of TonB protein in wild-type and protease deficient strains, (iv) to localize TonB protein more precisely within the cell envelope, and (v) to determine the molecular mechanism(s) by which tonB gene expression is regulated in response to iron limitation. Purified TonB protein will be used to prepare anti-TonB antisera in order to develop a quantitative assay for TonB protein in cell extracts. Recombinant DNA techniques will be used for the construction of strains that overproduce TonB protein, and for the construction of tonB-lacZ and tonB-tetA transcriptional fusion strains in order to study tonB regulation and to obtain tonB regulatory mutants. In vitro mutagenesis with sodium bisulfite and DNA sequence analysis will be used to isolate and characterize mutations in cis-acting regulatory elements. Transposon Tn5 mutagenesis will be used to isolate mutations in trans-acting regulatory elements. RNA-DNA hybridization experiments will be performed in order to determine levels of tonB RNA synthesis under different growth conditions. The proposed research will provide insight into the regulation and synthesis of bacterial envelope proteins, in general, and it will lead to a better understanding of the role of the tonB gene product in envelope functions and iron transport in particular. The growing awareness that the course of bacterial infection is determined, in part, by the availability of iron in the blood and in part by the ability of pathogenic microorganisms to sequester iron lends medical relevance to this proposal.